| Fascioliasis is a zoonotic parasitic disease caused by infection with Fasciola gigantica and Fasciola hepatica.It is estimated that the economic loss every year caused by fascioliasis was over 3billion dollars worldwide.In addition,fascioliasis has been listed by the World Health Organization(WHO)as one of the neglected tropical human diseases,at least 240 million people worldwide were infected with fascioliasis,and 180 million people are at risk of infection.Therefore,fascioliasis is an important public health problem affecting the world and the development of animal husbandry.In previous proteomic study of Fasciola,it was found that 14-3-3 epsilon protein isoform(14-3-3e)and Ras-related protein Rab-10-like(Rab-10)are excretory-secretory proteins of F.gigantica(FgESPs),and it can be combined with goat peripheral blood mononuclear cells(PBMCs)in vivo.By review of the literature we found that 14-3-3 family proteins are involved in a variety of biological processes of parasites,including primary metabolism,signal transduction,cell cycle regulation,protein transport,transcription and stress response.The Ras family protein is a key component of many signal transduction pathways and plays an important role in parasite growth and development.However,the effect of 14-3-3e and Rab-10 on the functions of goat PBMCs is still unknown.Therefore,our study aimed to clone and express F.gigantica 14-3-3e gene and Rab-10 gene,and explore the functional effects of recombinant 14-3-3e and Rab-10 after in vitro stimulation of goat PBMCs.By cloning,expression and sequence analysis of the 14-3-3e gene and Rab-10 gene of F.gigantica,the results showed that their sizes were 774 bp and 618 bp,encoding 257 and 205 amino acid residues,respectively,and the relative molecular weights were approximately 30 kDa and 23 kDa;Their sequences were 100%and 99%homologous to the 14-3-3 protein and Ras family protein of F.hepatica,respectively.The expression of recombinant 14-3-3e(r14-3-3e)was soluble,while recombinant Rab-10(rRab-10)was expressed as inclusion body,and both of them had good purification effect.Western-blot analysis using goat serum that had been experimentally infected with F.gigantica showed that both recombinant proteins had good immunogenicity.When two different concentrations of the two recombinant proteins were co-incubated with goat PBMCs,respectively,their incorporation with goat PBMCs was observed by indirect immunofluorescence(IFA),and the results showed that both recombinant proteins combined with goat PBMCs.The secretion of IL-4,IL-10,TGF-βand IFN-γin goat PBMCs was examined by enzyme-linked immunosorbent assay(ELISA),the results showed that r14-3-3e significantly inhibited the secretion of IL-4 and IFN-γin goat PBMCs,significantly promoted the secretion of IL-10 and TGF-β,while rRab-10 significantly promoted the secretion of IL-4,IL-10,TGF-βand IFN-γin goat PBMCs.The Cell Counting Kit-8(CCK-8)was used to detect the proliferation of goat PBMCs,the results showed that the two recombinant proteins significantly inhibited the proliferation of goat PBMCs.By using 6.5 mm Transwell~?with 8.0μm-pore-size polyester membrane inserts to test the migration of goat PBMCs,we found that r14-3-3e significantly inhibited the migration of goat PBMCs,while rRab-10 significantly promoted the migration of goat PBMCs.Total Nitric Oxide Assay Kit was used to detect the production of Nitric oxide(NO)in goat PBMCs,and the results showed that both recombinant proteins could significantly promote NO production in goat PBMCs.Flow cytometry was used to examine the phagocytic ability of goat monocytes,and the results showed that r14-3-3e significantly inhibited the phagocytic ability of goat monocytes,while rRab-10 significantly promoted the phagocytic ability of goat monocytes.Our study revealed that 14-3-3e and Rab-10 are two important ESPs of F.gigantica and can affect the immune response of the host.When they interacted with goat PBMCs,14-3-3e showed significant immunosuppressive effects,while Rab-10 showed significant immunopromoting effects.This study laid the foundation for further elucidation of the mechanism of immune evasion,immune interference and immunosuppression after F.gigantica infection. |
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